miR-338-5p通过靶向TSHZ3促进膀胱癌细胞增殖、迁移和侵袭

doi:10.11958/20221910

摘要/Abstract

摘要：

目的探讨miR-338-5p通过靶向TSHZ3调控膀胱癌细胞增殖、迁移和侵袭的机制。方法实时荧光定量PCR（qRT-PCR）检测miR-338-5p在33例膀胱癌组织和癌旁组织中的表达；在膀胱癌T24和UM-UC-3细胞中分别转染mimics-NC、miR-338-5p mimics、inhibitor-NC和miR-338-5p inhibitor，qRT-PCR检测转染效率；采用CCK-8实验检测过表达或者敲低miR-338-5p对膀胱癌细胞增殖能力的影响；Transwell实验检测miR-338-5p对膀胱癌细胞迁移和侵袭能力的影响；TargetScan、miRDB和Targetminer数据库预测miR-338-5p潜在的靶基因，选定靶基因TSHZ3；双萤光素酶报告基因实验和Western blot验证miR-338-5p和TSHZ3的靶向关系；在膀胱癌细胞中共转染miR-338-5p mimics和TSHZ3过表达质粒，通过CCK-8和Transwell实验检测细胞的增殖、迁移和侵袭能力；Western blot检测过表达miR-338-5p或者TSHZ3对Wnt/β-catenin信号通路的影响。结果 miR-338-5p在膀胱癌中表达上调（P<0.05）；过表达miR-338-5p提高了膀胱癌细胞的增殖、迁移和侵袭能力（P<0.05），转染miR-338-5p inhibitor则降低了膀胱癌细胞的增殖、迁移和侵袭能力（P<0.05）；生物信息学分析和双萤光素酶报告基因实验表明，miR-338-5p和TSHZ3存在靶向关系；挽救实验表明，同时过表达miR-338-5p和TSHZ3部分消除了过表达miR-338-5p对膀胱癌细胞增殖、迁移和侵袭能力的促进作用（P<0.05）；Western blot结果显示，与mimics-NC组比较，过表达miR-338-5p降低了TSHZ3蛋白表达水平，提高了Wnt3a和β-catenin蛋白表达水平（P<0.05）；过表达TSHZ3降低了Wnt3a和β-catenin蛋白表达水平（P<0.05）。结论 miR-338-5p在膀胱癌中表达上调，通过靶向抑制TSHZ3的表达促进膀胱癌的增殖、迁移和侵袭，该机制可能与激活Wnt/β-catenin信号通路有关。

关键词: 膀胱肿瘤, 细胞增殖, 细胞运动, 肿瘤浸润, miR-338-5p, Teashirt锌指同源盒3, Wnt/β-catenin

Abstract:

Objective To investigate the mechanism of miR-338-5p regulating the proliferation, migration and invasion of bladder cancer cells by targeting TSHZ3. Methods The expression of miR-338-5p in 33 samples of bladder cancer tissue and paracancerous tissue was detected by qRT-PCR. Bladder cancer T24 and UM-UC-3 cells were transfected with mimics-NC, miR-338-5p mimics, inhibitor-NC and miR-338-5p inhibitor, and the transfection efficiency was detected by qRT-PCR. CCK-8 assay was used to detect the effect of overexpression or knockdown of miR-338-5p on the proliferation of bladder cancer cells. Transwell assay was used to detect the effect of miR-338-5p on migration and invasion of bladder cancer cells. The potential target genes of miR-338-5p were predicted by TargetScan, miRDB and Targetminer databases, and TSHZ3 was chosen for the target gene. The targeting relationship between miR-338-5p and TSHZ3 was verified by dual-luciferase reporter gene assay and Western blot assay. miR-338-5p mimics and TSHZ3 overexpression plasmids were co-transfected in bladder cancer cells, and the proliferation, migration and invasion of cells were detected by CCK-8 and Transwell assays. Western blot assay was used to detect the overexpression of miR-338-5p or TSHZ3 against Wnt/ β-catenin signaling pathway. Results miR-338-5p was significantly upregulated in bladder cancer (P<0.05). Overexpression of miR-338-5p increased the proliferation, migration and invasion of bladder cancer cells (P<0.05), while transfection of miR-338-5p inhibitor decreased the proliferation, migration and invasion of bladder cancer cells (P<0.05). Bioinformatics analysis and the dual-luciferase reporter gene assay showed that there was a targeting relationship between miR-338-5p and TSHZ3. The rescue experiment showed that co-transfection of miR-338-5p mimics and TSHZ3 overexpression plasmid partially eliminated the promoting effect of overexpression of miR-338-5p on the proliferation, migration and invasion of bladder cancer cells (P<0.05). Western blot results showed that overexpression of miR-338-5p significantly decreased the expression level of TSHZ3 protein and increased Wnt3a and β-catenin protein expression levels (P<0.05). In addition, overexpression of TSHZ3 reduced Wnt3a and β-catenin protein expression levels (P<0.05). Conclusion miR-338-5p is upregulated in bladder cancer, and promotes the proliferation, migration and invasion of bladder cancer by targeting TSHZ3 expression. The mechanism may be related to the activation of Wnt/β-catenin signaling pathway.

Key words: urinary bladder neoplasms, cell proliferation, cell movement, neoplasm invasiveness, miR-338-5p, TSHZ3, Wnt/β-catenin

中图分类号:

R737.14

图/表 17

图1 miR-338-5p在正常输尿管上皮细胞和膀胱癌细胞中的表达 a表示与SV-HUC-1比较，P<0.05。

Fig.1 The expression of miR-338-5p in normal urothelial cells and bladder cancer cells

图2 过表达和敲低miR-338-5p的转染效率

Fig.2 Transfection efficiency of overexpression and inhibition of miR-338-5p

图3 CCK-8检测过表达miR-338-5p对细胞增殖的影响＊P<0.05，＊＊P<0.01，n=3；A：T24细胞培养第1、2、3、4和5天t值分别为0.131、1.526、2.887、7.560＊＊和6.876＊＊；B：UM-UC-3细胞培养第1、2、3、4和5天t值分别为0.670、2.793、3.019＊、7.479＊＊和8.758＊＊。

Fig.3 Effect of overexpression of miR-338-5p on cell proliferation detected by CCK-8 assay

图4 CCK-8检测抑制miR-338-5p表达对细胞增殖的影响＊P<0.05，＊＊P<0.01，n=3；A：T24细胞培养第1、2、3、4和5天t值分别为0.745、1.084、1.348、6.023＊＊和5.481＊＊；B：UM-UC-3细胞培养第1、2、3、4和5天t值分别为0.650、1.730、1.789、3.307＊和4.945＊＊。

Fig.4 Effect of inhibition of miR-338-5p on cell proliferation detected by CCK-8 assay

表1 各组迁移和侵袭细胞数比较 ( n=3,个/视野, $\bar{x}±s$)

Tab.1 Comparison of the number of migrated and invaded cells between the four groups

组别	T24
组别	迁移	侵袭
mimics-NC组	136.00±3.61	138.34±3.51
miR-338-5p mimics组	200.67±4.51	202.67±8.50
t	19.400^＊＊	12.110^＊＊
inhibitor-NC组	327.00±6.56	305.67±8.02
miR-338-5p inhibitor组	252.33±6.51	211.33±8.33
t	14.000^＊＊	14.132^＊＊
组别	UM-UC-3
组别	迁移	侵袭
mimics-NC组	237.00±5.57	291.67±7.02
miR-338-5p mimics组	387.67±5.13	468.33±3.06
t	34.645^＊＊	39.950^＊＊
inhibitor-NC组	344.33±5.03	375.00±9.00
miR-338-5p inhibitor组	146.00±7.21	159.67±4.51
t	39.064^＊＊	37.051^＊＊

图5 过表达miR-338-5p对膀胱癌细胞迁移和侵袭的影响（结晶紫染色，×200）

Fig.5 Effects of overexpression of miR-338-5p on migration and invasion of bladder cancer cells (crystal violet staining, ×200)

图6 抑制miR-338-5p表达对膀胱癌细胞迁移和侵袭的影响（结晶紫染色，×200）

Fig.6 Effects of inhibition of miR-338-5p on migration and invasion of bladder cancer cells (crystal violet staining, ×200)

图7 TSHZ3的3'-UTR与miR-338-5p互补的核苷酸序列

Fig.7 Nucleotide sequences complementary to miR-338-5p in the 3'-UTR of TSHZ3

表2 各组萤光素酶活性比较 (n=3, $\bar{x}±s$)

Tab.2 Comparison of the luciferase activity between the two groups

组别	T24		UM-UC-3
组别	TSHZ3-wt	TSHZ3-mut	TSHZ3-wt	TSHZ3-mut
mimics-NC组	1.00±0.25	0.94±0.05	1.06±0.07	1.02±0.11
miR-338-5p mimics组	0.54±0.07	0.91±0.03	0.49±0.04	0.96±0.04
t	10.473^＊＊	1.067	12.173^＊＊	0.888

图8 过表达miR-338-5p对TSHZ3 mRNA表达的影响

Fig.8 Effect of overexpression of miR-338-5p on TSHZ3 mRNA expression

图9 过表达miR-338-5p对TSHZ3蛋白水平的影响

Fig. 9 Effect of overexpression of miR-338-5p on TSHZ3 protein level

图10 TSHZ3 mRNA在正常输尿管上皮细胞和膀胱癌细胞中的表达

Fig.10 The mRNA expression of TSHZ3 in normal urothelial cells and bladder cancer cells

图11 CCK-8检测各组细胞增殖活力＊P<0.05，＊＊P<0.01，n=3；A：T24细胞培养第1、2、3、4和5天F值分别为0.157、6.025＊、28.297＊＊、58.467＊＊和35.961＊＊；B：UM-UC-3细胞培养第1、2、3、4和5天F值分别为1.049、2.120、16.651＊＊、35.161＊＊和40.433＊＊。

Fig.11 Cell viability detected by CCK-8 assay in each group

表3 各组迁移和侵袭细胞数比较 (n=3, 个/视野, $\bar{x}±s$)

Tab.3 Comparison of the number of migrated and invaded cells between the three groups

组别		T24
组别		迁移	侵袭
mimics-NC组		144.33±4.51	151.00±6.00
miR-338-5p mimics组		309.67±5.51^a	356.33±8.50^a
miR-338-5p mimics+TSHZ3组		204.67±7.02^b	214.67±9.50^b
F		630.003^＊＊	500.471^＊＊
组别	UM-UC-3
组别	迁移		侵袭
mimics-NC组	173.67±5.03		101.00±6.00
miR-338-5p mimics组	332.67±4.73^a		284.33±6.51^a
miR-338-5p mimics+TSHZ3组	255.00±7.94^b		213.00±6.56^b
F	514.087^＊＊		633.505^＊＊

图12 各组细胞迁移侵袭情况（结晶紫染色，×200）

Fig.12 Migration and invasion of each group of cells (crystal violet staining, ×200)

图13 过表达miR-338-5p对Wnt3a和β-catenin蛋白水平的影响

Fig.13 Effect of overexpression of miR-338-5p on the protein level of Wnt3a and β-catenin

图14 过表达TSHZ3对Wnt3a和β-catenin蛋白水平的影响

Fig.14 Effect of overexpression of TSHZ3 on the protein level of Wnt3a and β-catenin

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